Influence of cell-adhesive peptide ligands on poly(ethylene glycol) hydrogel physical, mechanical and transport properties

Abstract

Synthetic three-dimensional scaffolds for cell and tissue engineering routinely utilize peptide ligands to provide sites for cell adhesion and to promote cellular activity. Given the fact that recent studies have dedicated great attention to the mechanisms by which cell behavior is influenced by various ligands and scaffold material properties, it is surprising that little work to date has been carried out to investigate the influence of covalently bound ligands on hydrogel material properties. Herein we report the influence of three common ligands utilized in tissue engineering, namely RGD, YIGSR and IKVAV, on the mechanical properties of cross-linked poly(ethylene glycol) (PEG) hydrogels. The effect of the ligands on hydrogel storage modulus, swelling ratio, mesh size and also on the diffusivity of bovine serum albumin through the hydrogel were investigated in detail. We identified conditions under which these ligands strikingly influence the properties of the material. The extent of influence and whether the ligand increases or decreases a specific property is linked to ligand type and concentration. Further, we pinpoint mechanisms by which the ligands interact with the PEG network. This work thus provides specific evidence for interactions between peptide ligands and cross-linked PEG hydrogels that have a significant impact on hydrogel material and transport properties. As a result, this work may have important implications for interpreting cell experiments carried out with ligand-modified hydrogels, because the addition of ligand may affect not only the scaffold's biological properties, but also key physical properties of the system.

Figure 1

Schematic of the Michael-type addition reaction to form PEG hydrogels with covalently incorporated ligands. In the first step (a), 4-arm PEG-VS is functionalized with adhesive ligands via an unpaired cysteine residue. The ligand is added at a large stoichiometric deficit to VS groups. In a second cross-linking step (b), a PEG-dithiol cross-linker is added such that total VS/SH = 1:1 to form a 3D hydrogel network.

Figure 2

PEG hydrogel with covalently incorporated fluorescent ligand was used to determine that unbound ligand: a) was released in the supernatant PBS initially prior to 4 h, but b) was not released from the hydrogel over time (i.e., differences between sample fluorescence at time points up to 72 h are not statistically distinct).

Figure 3

a) Schematic representation of hydrogel mesh disruption upon covalent binding of ligand or PEG-SH to the 4-arm PEG-VS; b) Influence of ligand type on PEG hydrogel swelling ratio (Q_M). All hydrogels were prepared as 10% w/v polymer with 100 μM ligand. Asterisks designate significant differences.

Figure 4

Influence of ligand type on PEG hydrogel swelling ratio (Q_M). Each plot highlights a different ligand property: a) net charge; b) hydrophobicity index; c) pI. All hydrogels were prepared as 10% w/v polymer with 100 μM ligand.

Figure 5

a) Influence of ligand type on PEG hydrogel mesh size; b) Influence of ligand type on PEG hydrogel storage modulus (G′). All hydrogels were prepared as 10% w/v polymer with 100 μM ligand. Asterisks designate significant differences.

Figure 6

a) Schematic representation of hydrogen bonding between the phenolic OH group of the tyrosine (Y) amino acid of the YIGSR and YIGSRPD ligands and the ether oxygen of the PEG polymer; b) Influence of buffer pH on the swelling ratios of PEG hydrogels containing no ligand or 100 μM YIGSR. All hydrogels were 10% w/v. Asterisks designate significant differences.

Figure 7

Influence of ligand concentration on swelling ratio (Q_M) of PEG hydrogels: a) RGDS ligand; b) IKVAV ligand; c) YIGSR ligand; d) PEG-SH control. All hydrogels were prepared as 10% w/v polymer. Asterisks designate significant differences.

Figure 8

Influence of ligand concentration on mesh size of PEG hydrogels: a) RGDS ligand; b) IKVAV ligand; c) YIGSR ligand; d) PEG-SH control. All hydrogels were prepared as 10% w/v polymer. Asterisks designate significant differences.

Figure 9

Influence of ligand concentration on storage modulus (G′) of PEG hydrogels: a) RGDS ligand; b) IKVAV ligand; c) YIGSR ligand; d) PEG-SH control. All hydrogels were prepared as 10% w/v polymer. Asterisks designate significant differences.

Figure 10

Comparison between PEG hydrogels with varying polymer density prepared with 100 μM YIGSR ligand or no ligand and the effects on: a) swelling ratio, b) mesh size, c) and storage modulus. Asterisks designate significant differences.

Figure 11

Effect of ligand type on BSA diffusivity (D_e) in PEG hydrogels. All hydrogels were prepared as 10% w/v polymer with 100 μM ligand. Asterisks designate significant differences. The BSA diffusivity in the hydrogel is normalized by that of BSA diffusivity in water (D_o = 0.941×10⁻⁵ mm²/s).